Engineering Success | SubCat-HS

Parkinson's disease (PD) is one of the common diseases in neurodegenerative diseases, so the early diagnosis of PD can effectively prevent the development process [1]. The pathophysiology of PD manifest as the loss of neurons in locus coeruleus and substantia nigra [2]. Studies have shown that PD pathogenesis is related to the accumulation of α-synuclein (α-syn) in substantia nigra, and the expression level of α-syn in cerebrospinal fluid can be used as an important specific biomarker for early diagnosis of PD [3-4]. Besides α-syn, Aβ42 show highly relevant in PD, because some PD patients exhibit amyloid plaques similar to those found in Alzheimer's diseases [5]. NF-L is a neuronal structural protein, which represents a key biomarker of nerve damage when its expression level elevating [5]. NF-L provides a quantitative indicator to track neurodegeneration [5]. Therefore, our project intends to obtain PD-specific markers NF-L, Aβ42, and α-syn by genetic engineering methods, and then verifies the accuracy of recombination proteins by ELISA assay. Finally, we expect to prepare for the acquisition of specific antibodies and the construction of early PD diagnosis kits.

Design:

The pET28a(+) plasmid was provided by the strain library of our unit, and the target gene NF-L was synthesized by a biotechnology company. The NF-L fragment was homologously recombined with the linearized plasmid to construct pET28a-NF-L.

Fig 1. The plasmid map of pET28a-NF-L

Build:

We constructed pET28a-NF-L by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp. Figure 2B shows a band consistent with the target size. The NF-L gene was amplified by PCR, with a length of 1059 bp. Figure 2A shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained by homologous recombination.

Fig. 2 Agarose gel electrophoresis of target genes and linearized pET-28a(+) vector

The recombinant plasmid was introduced into E. coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 3B shows that the length is consistent with the target fragment, Figure 3A is the clone on the plate, and the results in Figure 4 show that the NF-L gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a-NF-L was successfully constructed.

Fig. 3 Verification of the transformation results of pET-28a(+)-NF-L/Aβ-42/α-syn positive colony. A: The single colony on the plate; B: The amplification results of colony PCR.

Fig. 4 Sanger sequencing results of pET-28a(+)-NF-L/Aβ-42/α-syn positive colony.

Design:

The pET28a(+) plasmid was provided by the strain library of our unit, and the target gene Aβ42 was synthesized by a biotechnology company. The Aβ42 fragment was homologously recombined with the linearized plasmid to construct pET28a-Aβ42.

Fig 5. The plasmid map of pET28a-Aβ42

We constructed pET28a-Aβ42 by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp. Figure 2B shows a band consistent with the target size. The NF-L gene was amplified by PCR, with a length of 789 bp. Figure 2A shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. The recombinant plasmid was obtained by homologous recombination after agarose gel electrophoresis and gel recovery.

The recombinant plasmid was introduced into E. coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 3B shows that the length is consistent with the target fragment, Figure 3A is the clone on the plate, and the results in Figure 4 show that the Aβ42 gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a- Aβ42 was successfully constructed.

Design:

The pET28a(+) plasmid was provided by the strain library of our unit, and the target gene α-syn was synthesized by a biotechnology company. The α-syn fragment was homologously recombined with the linearized plasmid to construct pET28a-α-syn.

Fig 6. The plasmid map of pET28a-α-syn

Build:

We constructed pET28a-α-syn by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp. Figure 2B shows a band consistent with the target size. The NF-L gene was amplified by PCR, with a length of 522 bp. Figure 2A shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. The recombinant plasmid was obtained by homologous recombination after agarose gel electrophoresis and gel recovery.

The recombinant plasmid was introduced into E. coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 3B shows that the length is consistent with the target fragment, Figure 3A is the clone on the plate, and the results in Figure 4 show that the α-syn gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a- α-syn was successfully constructed.

Design:

The pET28a(+) plasmid and mCherry gene were provided by the strain library of our unit, and the target gene NF-L was synthesized by a biotechnology company. The mCherry fragment was homologously recombined with the NF-L fragment and the linearized plasmid to construct pET28a-NF-L-mCherry.

Fig 7. The plasmid map of pET28a-NF-L-mChaerry

Build:

We constructed pET28a-NF-L-mCherry by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp, and Figure 2B shows a band consistent with the target size. The NF-L gene was amplified by PCR, with a length of 1059 bp, and Figure 8A shows a band consistent with the target size. The mCherry gene was amplified by PCR, with a length of 711 bp, and Figure 8B shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained by homologous recombination.

Fig. 8 Agarose gel electrophoresis of target genes and mcherry fragment

The recombinant plasmid was transferred into Escherichia coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 9B shows that the length is consistent with the target fragment, Figure 9A is the clone on the plate, and the results in Figure 10 show that the NF-L and mCherry gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a-NF-L- mCherry was successfully constructed.

Fig. 9 Verification of the transformation results of pET-28a(+)-NF-L-mcherry/Aβ-42-mcherry /α-syn-mcherry positive colony. A: The single colony on the plate; B: The amplification results of colony PCR.

Fig. 10 Sanger sequencing results of pET-28a(+)-NF-L-mcherry/Aβ-42-mcherry /α-syn-mcherry positive colony.

Design:

The pET28a(+) plasmid and mCherry gene were provided by the strain library of our unit, and the target gene Aβ42 was synthesized by a biotechnology company. The mCherry fragment was homologously recombined with the Aβ42 fragment and the linearized plasmid to construct pET28a- Aβ42-mCherry.

Fig 11. The plasmid map of pET28a- Aβ42-mCherry

We constructed pET28a- Aβ42-mCherry by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp, and Figure 2B shows a band consistent with the target size. The Aβ42 gene was amplified by PCR, with a length of 789 bp, and Figure 8A shows a band consistent with the target size. The mCherry gene was amplified by PCR, with a length of 711 bp, and Figure 8B shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained by homologous recombination.

The recombinant plasmid was transferred into Escherichia coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 9B shows that the length is consistent with the target fragment, Figure 9A is the clone on the plate, and the results in Figure 10 show that the Aβ42 and mCherry gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a- Aβ42- mCherry was successfully constructed.

Design:

The pET28a(+) plasmid and mCherry gene were provided by the strain library of our unit, and the target gene α-syn was synthesized by a biotechnology company. The mCherry fragment was homologously recombined with the α-syn fragment and the linearized plasmid to construct pET28a- α-syn -mCherry.

Fig 12. The plasmid map of pET28a- α-syn -mCherry

We constructed pET28a- α-syn -mCherry by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp, and Figure 2B shows a band consistent with the target size. The α-syn gene was amplified by PCR, with a length of 522 bp, and Figure 8A shows a band consistent with the target size. The mCherry gene was amplified by PCR, with a length of 711 bp, and Figure 8B shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained by homologous recombination.

The recombinant plasmid was transferred into Escherichia coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 9B shows that the length is consistent with the target fragment, Figure 9A is the clone on the plate, and the results in Figure 10 show that the α-syn and mCherry gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a- α-syn -mCherry was successfully constructed.

Test:

1. Protein expression

Based on the lac operator system in pET-28a(+) vector backbone, we used IPTG for protein prokaryotic expression. First, we used a final concentration of 1 mM/L IPTG for usual protein expression. Then, we set two temperature and different expression time for determining the optimal induction condition. The expected molecular weights of 6xHis-NF-L, 6xHis-NF-L-mcherry, 6xHis-Aβ-42, 6xHis-Aβ-42-mcherry, 6xHis-α-syn, and 6xHis-α-syn-mcherry are 44.8 kDa, 71.5 kDa, 34.0 kDa, 60.7 kDa, 23.3 kDa, and 50.0 kDa, respectively. As showed in Fig. 7, we can clearly observe protein bands of target genes. Interestingly, we found that the molecular weight of 6xHis-Aβ-42 and 6xHis-Aβ-42-mcherry are slightly larger than as predicted, suggesting that these two proteins might have some post-translational modifications. For 6xHis-NF-L and 6xHis-NF-L-mcherry, there was no significant difference in the bands between 25°C and 37°C, and the protein expression level was highest after 6 hours of induction. For 6xHis-Aβ-42 and 6xHis-Aβ-42-mcherry, there was no significant difference in the bands between 25°C and 37°C, and the protein expression level showed similar between after 3 hours and 6 hours induction, indicating that the protein synthesis rate had reached a plateau. Interestingly, 6xHis-α-syn showed different protein synthesis trends under 25°C and 37°C. Under 25°C, the protein expression level of 6xHis-α-syn was highest after 1 hours of induction, but as the induction time increased, the protein was further degraded. For 37°C, the protein expression level of 6xHis-α-syn was highest after 6 hours of induction. For 6xHis-α-syn-mcherry, the protein synthesis rate under 37°C is faster than that under 25°C, and the protein expression level was highest after 6 hours of induction.

Fig. 13 SDS-PAGE analysis for small amount of target proteins expression.

We further used anti-His antibody for Western Blotting experiments and then quantified the induction amount of target proteins according to the relative gray value of the band. As showed in Fig. 14A, we found that the His antibody specificity is not particularly high, suggesting that we might need to optimize the relative concentrations of the primary and secondary antibodies, but we can still clearly see the bands of target proteins. Then, we used ImageJ software to calculate the relative gray value intensity of target proteins. The results were consistent with those of Coomassie Brilliant Blue staining in Fig. 14. Different temperatures have an effect on the correct folding and biological activity of proteins. Finally, except for 6xHis-α-syn, we chose 25°C and 37°C after 6 hours of induction for large-scale purification. For 6xHis-α-syn, we chose 25°C after 1 hours of induction and 37°C after 6 hours of induction for large-scale purification, respectively.

Fig. 14 Western Blotting analysis and the relative gray value intensity for small amount of target proteins expression.

2. Protein His-tag purification

As showed in Fig. 15, we could clearly observe that the combination proteins with mcherry fluorescent tag have visible red precipitate. We further used His-tag purification kit to purify target proteins and then stained protein bands by Coomassie Brilliant Blue staining. As showed in Fig. 16 and Fig. 17, target proteins were enriched in elution components. Compared with proteins induced under 25°C, proteins induced under 37°C had better purification efficiency and less miscellaneous bands. In addition, we also found that the efficiency of protein binding with Ni-NTA beads is not particularly high, so we might need to optimize the conditions to obtain higher recovery yield. Then, we collected elution components for functional test.

Fig. 15 Representative images of large-scale protein expression.

Fig. 16 SDS-PAGE analysis for large-scale His-tag protein purification of target proteins expressed under 25°C. T: Total protein; FT: Flow Through.

Fig. 17 SDS-PAGE analysis for large-scale His-tag protein purification of target proteins expressed under 37°C. Total protein; FT: Flow Through.

2.1 Determination of protein fused with mcherry tag by fluorescence microscope

First, we determined the effectivity of target protein fused with mcherry tag by fluorescence microscope. After fixing sample and adding anti-fading agent, we detected mcherry signal using an excited light wavelength of 587nm and an emitted light wavelength of 610nm. As showed in Fig. 18, E.coli BL21 expressing pET-28a(+)-NF-L-mcherry/Aβ-42-mcherry/α-syn-mcherry could be clearly observed with red fluorescence in cord-like structure.

Fig. 18 Observation of E.coli BL21 expressing pET-28a(+)-NF-L-mcherry/Aβ-42-mcherry/α-syn-mcherry by fluorescence microscopy

2.2 Verification of target recombination protein by ELISA assay

We used ELISA assay to verify the accuracy of NF-L protein. As showed in Fig. 19A, the correlation between NF-L protein and absorbance 450nm is 0.9972, indicating that ELISA assay can accurately and effectively detect the content of NF-L in elution components. In elution components of protein expression under 25°C, the content of NF-L protein in E6 was the highest, reaching 25.31 ng/mL (Fig. 19B). Interestingly, in elution components of protein expression under 37°C, the content of NF-L protein in E2 was the highest, reaching 34.25 ng/mL (Fig. 19C).

Fig. 19 Verification of NF-L by ELISA assay

Then, we used ELISA assay to verify the accuracy of Aβ-42 protein. As showed in Fig. 20A, the correlation between Aβ-42 protein and absorbance 450nm is 0.9983, indicating that ELISA assay can accurately and effectively detect the content of Aβ-42 in elution components. In elution components of protein expression under 25°C, the content of Aβ-42 protein in E6 was the highest, reaching 21.35 pg/mL (Fig. 20B). In elution components of protein expression under 37°C, the content of Aβ-42 protein in E6 was the highest, reaching 26.92 pg/mL (Fig. 20C).

Fig. 20 Verification of Aβ-42 by ELISA assay

Finally, we used ELISA assay to verify the accuracy of α-syn protein. As showed in Fig. 21A, the correlation between α-syn protein and absorbance 450nm is 0.994, indicating that ELISA assay can accurately and effectively detect the content of α-syn in elution components. In elution components of protein expression under 25°C, the content of α-syn protein in E6 was the highest, reaching 97.54 ng/mL (Fig. 21B). In elution components of protein expression under 37°C, the content of α-syn protein in E6 was the highest, reaching 98.32 ng/mL (Fig. 21C).

Fig. 21 Verification of α-syn by ELISA assay

Our experiment did not optimize the protein expression conditions, and fermentation regulation plays a crucial role in yield. We can continue to optimize the expression conditions of the strains, such as oxygen levels, inoculum size, and pH, to improve production and to achieve the amount of protein required for antigen preparation.

In our expression process, we added inducers IPTG. We can explore more environmentally friendly production methods by modifying promoter of vector backbone or replacing the inducers with external regulatory conditions, such as temperature and light exposure, to induce expression through these greener and harmless approaches.

Goedert M. Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci. (2001) 2:492–501.

Braak H, Ghebremedhin E, Rüb U, Bratzke H, Del Tredici K. Stages in the development of Parkinson's disease-related pathology. Cell Tissue Res. (2004) 318(1):121-34.

Giasson BI, Murray IV, Trojanowski JQ, Lee VM, A. hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. J Biol Chem. (2001) 276:2380–6.

Hoyer W, Cherny D, Subramaniam V, Jovin TM. Impact of the acidic C-terminal region comprising amino acids 109-140 on alpha-synuclein aggregation in vitro. Biochemistry. (2004) 43:16233–42.

Xiang C, Cong S, Tan X, Ma S, Liu Y, Wang H, Cong S. A meta-analysis of the diagnostic utility of biomarkers in cerebrospinal fluid in Parkinson's disease. NPJ Parkinsons Dis. (2022) 29;8(1):165.